CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) is the target of mutations that cause cystic fibrosis (CF). CF is characterized by abnormal endocrine and exocrine gland function. In CF, unusually thick mucus leads to chronic pulmonary disease and respiratory infections, insufficient pancreatic and digestive function, and abnormally concentrated sweat. Seventy percent of the mutant CFTR alleles in the Caucasian population result from deletion of phenylalanine at position 508 (ΔF508-CFTR), the result of a three base pair deletion in the genetic code. Other mutations have also been described, e.g., a glycine to aspartate substitution at position 551 (G551D-CFTR) occurs in approximately 3-4% of cystic fibrosis patients.
The ΔF508-CFTR mutation results in a CFTR protein capable of conducting chloride, but absent from the plasma membrane because of aberrant intracellular processing. Under usual conditions (37° C.), the ΔF508-CFTR protein is retained in the endoplasmic reticulum (ER), by prolonged association with the ER chaperones, including calnexin and hsp70. Over expression of ΔF508-CFTR can result in ΔF508-CFTR protein appearing at the cell surface, and this protein is functional once it reaches the cell surface. The ΔF508-CFTR “trafficking” block is also reversible by incubation of cultured CF epithelial cells at reduced temperatures (25-27° C.). Lowered temperature results in the appearance of CFTR protein and channel activity at the cell surface, suggesting an intrinsic thermodynamic instability in ΔF508-CFTR at 37° C. that leads to recognition of the mutant protein by the ER quality control mechanism, prevents further trafficking, and results in protein degradation. Chemical chaperones are currently being developed to restore the folding of ΔF508-CFTR. However, when ΔF508-CFTR is expressed at the cell-surface following treatment, CAL (also known as CFTR-associated ligand, PIST, GOPC, ROS, and FIG) directs the lysosomal degradation of CFTR in a dose-dependent fashion and reduces the amount of CFTR found at the cell surface. Conversely, NHERF1 and NHERF2 functionally stabilize CFTR. Consistent with this role of CAL, RNA interference targeting of endogenous CAL also increases cell-surface expression of the disease-associated ΔF508-CFTR mutant and enhances transepithelial chloride currents in a polarized human patient bronchial epithelial cell line (Wolde, et al. (2007) J. Biol. Chem. 282:8099-8109).
Current treatments for cystic fibrosis generally focus on controlling infection through antibiotic therapy and promoting mucus clearance by use of postural drainage and chest percussion. However, even with such treatments, frequent hospitalization is often required as the disease progresses. New therapies designed to increase chloride ion conductance in airway epithelial cells have been proposed, and restoration of the expression of functional CFTR at the cell surface is considered a major therapeutic goal in the treatment of cystic fibrosis, a disease that affects˜30,000 patients in the U.S., and ˜70,000 patients worldwide. For example, KALYDECO (Ivacaftor; VX-770) is an FDA-approved compound that ‘potentiates’ the open probability (Po) of CFTR channels, including the G551D mutant, and thus ameliorates the underlying molecular lesion in this group of patients. A 48-week clinical trial showed excellent efficacy, including a 10.6% improvement in lung function (predicted forced expiratory volume in 1 second; FEV1), a 55% drop in pulmonary exacerbations, and a 48 mEq/L reduction in sweat chloride (Ramsey, et al. (2011) N. Engl. J. Med. 365:1663-72). While showing efficacy in subjects with the G551D mutation, KALYDECO is not useful as a monotherapy for the largest group of CF patients. In ˜70% of mutant alleles, Phe508 is deleted (ΔF508; Kerem, et al. (1989) Science 245: 1073-1080). As a result, ˜50% of CF patients are ΔF508 homozygous and ˜40% are heterozygous. Unfortunately, clinical trials in ΔF508 homozygotes show low efficacy for KALYDECO alone (Flume, et al. (2012) Chest 142:718-724).
In the absence of interventions, ΔF508-CFTR exhibits three defects: folding, gating, and stability (Riordan (2008) Annu. Rev. Biochem. 77:701-726; Cheng, et al. (1990) Cell 63:827-834; Lukacs, et al. (1993) J. Biol. Chem. 268:21592-21598; Dalemans, et al. (1991) Nature 354:526-528). However, if folding is restored, ΔF508-CFTR retains some channel activity (Drumm, et al. (1991) Science 254:1797-1799; Denning, et al. (1992) Nature 358:761-764). ‘Corrector’ compounds have been identified such as corr-4a (Pedemonte et al. (2005) J. Clin. Invest. 115:2564) and Lumacaftor (VX-809), which partially alleviate the folding defect and allows some ΔF508-CFTR to reach the apical membrane (Van Goor, et al. (2009) Pediatr. Pulmonol. 44:S154-S155; Van Goor, et al. (2011) Proc. Natl. Acad. Sci. USA 108:18843-18848). Although Lumacaftor yields only limited benefits in monotherapy, it shows greater efficacy in combination with KALYDECO: 25% of patients showed a >10% increase in FEV1 and 55% of patients showed >5% increase, with few adverse effects. While a 5% or 10% improvement is clinically meaningful, FEV1 drops approximately 1-2% per year in CF patients (Dasenbrook, et al. (2008) Am. J. Respir. Crit. Care Med. 178:814-821; Que, et al. (2006) Thorax 61:155-157), even in the absence of acceleration by pulmonary exacerbations (Taylor-Robinson, et al. (2012) Thorax 67:860-866). Thus, further improvements are required, especially for non-responders and the 40% of ΔF508-CFTR heterozygous patients.